This invention is concerned with applications of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for excitatory amino acid (EAA) receptors, especially human EAA receptors.
In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the xe2x80x9csendingxe2x80x9d neuron which then binds to a surface receptor on the xe2x80x9creceivingxe2x80x9d neuron, to cause excitation thereof. L-glutamate is the most abundant neurotransmitter in the CNS, and mediates the major excitatory pathway in vertebrates. Glutamate is therefore referred to as an excitatory amino acid (EAA) and the receptors which respond to it are variously referred to as glutamate receptors, or more commonly as EAA receptors.
Using tissues isolated from mammalian brain, and various synthetic EAA receptor agonists, knowledge of EAA receptor pharmacology has been refined somewhat. Members of the EAA receptor family can be grouped into three main types based on differential binding to such agonists. One type of EAA receptor, which in addition to glutamate also binds the agonist NMDA (N-methyl-D-aspartate), is referred to as the NMDA type of EAA receptor. Two other glutamate-binding types of EAA receptor, which do not bind NMDA, are named according to their preference for binding with two other EAA receptor agonists, namely AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate (2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate). Accordingly, receptors which bind glutamate but not NMDA, and which bind with greater affinity to kainate than to AMPA, are referred to as kainate type EAA receptors. Similarly, those EAA receptors which bind glutamate but not NMDA, and which bind AMPA with greater affinity than kainate are referred to as AMPA type EAA receptors.
The glutamate-binding EAA receptor family is of great physiological and medical importance. Glutamate is involved in many aspects of long-term potentiation (learning and memory), in the development of synaptic plasticity, in epileptic seizures, in neuronal damage caused by ischemia following stroke or other hypoxic events, as well as in other forms of neurodegenerative processes. The development of therapeutics which modulate these processes has been very difficult, due to the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of the EAA receptor. The brain derived tissues currently used to screen candidate drugs are heterogeneous receptor sources, possessing on their surface many receptor types which interfere with studies of the EAA receptor/ligand interface of interest. The search for human therapeutics is further complicated by the limited availability of brain tissue of human origin. It would therefore be desirable to obtain cells that are genetically engineered to produce only the receptor of interest. With cell lines expressing cloned receptor cDNA, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs.
Non-human cDNAs which appear to encode the NMDA-type of EAA receptor have recently been identified and isolated. A cDNA encoding a subunit polypeptide of an NMDA receptor in rat, designated NR1, has been isolated as described by Moriyoshi et al. in Nature 354: 31, 1991. This work has been extended to demonstrate six isoforms of NR1, presumably generated by combinations of alternative RNA splicing in the amino- and carboxy-terminal regions of NR1 (Anantharam et al. FEBS Lett. 305: 27, 1992; Durand et al. Proc. Natl. Acad. Sci. USA 89: 9359, 1992; Nakanishi et al. Proc. Natl. Acad. Sci. USA 89: 8552, 1992; Sugihara et al. Biochem. Biophys. Res. Commun. 185: 826, 1992). DNA encoding NR1 and one of its isoforms have also been cloned from mouse brain by Yamazaki et al. as described in FEBS Lett. 300: 39, 1992. Other rat NMDA receptor subunits, designated NR2A, NR2B and NR2C, have also been identified (Monyer et al. Science 256: 1217, 1992), as well as mouse NMDA receptor subunits which have been designated xcex51, xcex52 and xcex53 (Meguro et al. Nature 357: 70, 1992 and Kutsuwada et al. Nature 358: 36, 1992).
There has emerged from these molecular cloning advances, a better understanding of the structural features of NMDA receptors and their subunits, as they exist in the non-human brain. According to the current model, each NMDA receptor is heteromeric, consisting of individual membrane-anchored subunits, each with four transmembrane regions, and extracellular domains that dictate ligand-binding properties and contribute to the ion-gating function served by the receptor complex.
In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable to obtain knowledge of human NMDA-type EAA receptors. A specific understanding of these human receptors would provide a means to screen for compounds that react therewith, i.e. to stimulate or inhibit receptor activity, and thus providing a means to identify compounds having potential therapeutic utility in humans. Non-human mammalian models are not suitable for this purpose despite significant receptor sequence homology, as minute sequence discrepancies can cause dramatic pharmacological variation between species homologues of the same receptor (Oksenberg et al., Nature, 360:161, 1992). It is therefore particularly desirable to provide cloned cDNA encoding human EAA receptors, and cell lines expressing these receptors in a homogeneous fashion, in order to generate a screening method for compounds therapeutically useful in humans. These, accordingly, are objects of the present invention.
Another object of the present invention is to provide in isolated form a DNA molecule which codes for a human EAA receptor.
It is another object of the present invention to provide a cell that has been genetically engineered to produce an N-methyl-D-aspartate-type human EAA receptor.
Human cDNAs encoding a family of EAA receptors, which bind glutamate with an affinity typical of EAA receptors and exhibit ligand binding properties characteristic of NMDA-type EAA receptors, have been identified and characterized. A representative member of this human EAA receptor family is herein designated human NMDAR1-1. Sequence-related cDNAs encoding naturally occurring variants of the human NMDAR1-1 have also been identified, and constitute additional members of this receptor family as do fragments of NMDAR1 receptors, herein referred to as the human NMDAR1 receptor family.
The present invention thus provides, in one of its aspects, an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human NMDAR1 or for fragments thereof characterized by at least one of MK-801-binding or glutamate-binding.
In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a human EAA receptor belonging to the herein-defined NMDAR1 family. In related aspects of the present invention, there are provided recombinant DNA constructs and relevant methods useful to create such cells.
In another aspect of the present invention, there is provided a method for evaluating interaction between a test ligand and a human EAA receptor, which comprises the steps of incubating the test ligand with a genetically engineered cell of the present invention, or with a membrane preparation derived therefrom, and then assessing said interaction by determining receptor/ligand binding.
Other aspects of the present invention, which encompass various applications of the discoveries herein described, will become apparent from the following detailed description, and from the accompanying drawings in which: